The long-term goal of this project is to understand beta-sheet folding energetics at a level that enables protein and proteomimetic design for therapeutic purposes. Comprehending beta-sheet folding energetics and contrasting them with misfolding energetics is essential for understanding the role of protein folding in health and disease, and for designing backbone modified proteins with enhanced physical properties. In specific aim 1 (50% effort), we employ a backbone and side chain mutagenesis strategy to test the hypothesis that a few energetically key residues make context-dependent, synergistic, hydrophobic and H-bonding contributions to native state stability. Such residues may be identifiable a priori owing to their extent of solvent exposure. Furthermore, we test the idea that backbone amide transfer free energies may be sensitive to the size and type of flanking side chains;if so this feature can easily be factored into beta-sheet design. In addition, we will explore the possibility of replacing pairs of solvent shielded backbone amides that make stabilizing H-bonds with E-olefin dipeptide isosteres to discern whether a backbone- backbone hydrophobic interaction could replace one or more H-bonds. This is the first step towards designing and synthesizing proteomimetics that fold and function despite having significantly fewer amide bonds than their ribosomally derived counterparts. Such mimetics could have substantially better membrane permeability properties than standard proteins, perhaps enabling aerosol and oral bioavailability. In specific aim 2 (50% effort), we take on the challenging task of understanding how N-glycosylation influences beta-sheet folding energetics, so that we can integrate this information into protein folding and secretion models that also consider the role of chaperones, folding enzymes, and degradation machinery. Any factor that influences protein folding kinetics and thermodynamics is likely to affect the partitioning of proteins between degradation pathways and normal folding and trafficking pathways. We test several hypotheses, including the idea that N-glycosylation of asparagine residues influences beta-sheet folding kinetics and thermodynamics, that a phi-value analysis as a function of oligosaccharide structure will reveal molecular details of the mechanism by which N-glycosylation influences folding, and that multiple glycosylation sites collaborate to influence folding of immunoglobulin domains, the fold found in antibody drugs.